This invention relates generally to urea assay methodology and, more particularly, to a colorimetric urea nitrogen reagent and method employing same.
Essentially three groups of methods have been used for the determination of urea nitrogen (UN). They can be classified as miscellaneous methods, methods based on the reaction with diacetyl-monoxime or similar compounds, and methods employing urease. Methods employing diacetyl-monoxime and urease have become the most popular.
In 1939, Fearon [Biochem. J. 33:902-907 (1930)] found that the reaction of diacetyl monoxime (DAM) followed by oxidation produces colors with R.sub.1 NH--CO--NHR.sub.2 when R.sub.1 is either H or a single aliphatic radical. R.sub.2 is not an acyl radical and is usually hydrogen. Colors were produced with urea, creatinine, methylurea (and urea derivatives), allantoin, and proteins. Many urea substitutes yield a red pigment, but only urea produces a yellow color. [Koritz et al, J. Bio. Chem. 209:145-150 (1954)].
In 1942, Ormbsy [J. Biol. Chem. 146:595-604 (1942)] applied the diacetyl-monoxime (DAM) reaction in a strongly acidic medium to the determination of urea. The resultant color was intensified by oxidation with potassium persulfate. Other oxidants have been used and are also described in the literature.
Natelson, Scott and Beffa [Am. J. Clin. Pathol. 21:275-281 (1951)] obviated the need for oxidizing agents by use of free diacetyl rather than the derivative.
In 1963, Coulombe and Favreau [Clin. Chem. 9:102-108 (1963)] demonstrated the effect of thiosemicarbazide upon the direct reaction between diacetyl-monoxime and urea. This compound further intensified the color and inhibits photosensitivity. Subsequently, the semi-carbazide reagent was incorporated in the automated procedure described by Pellerin [Clin. Chem. 10:374-375 (1964)].
In 1965, Marsh, Fingerhut, and Miller [Clin. Chem. 11:624-627 (1965)] presented both an automated and a manual diacetyl-monoxime method for direct determination of urea. Both methods proved highly sensitive due to the combined use of thiosemicarbazide and ferric ions.
In spite of the above advances, such urea determinations have been plagued by instability of working solutions resulting in limited shelf life and potentially erroneous results.